Produção e purificação integrada de protease fibrinolítica de Mucor subtilissimus UCP 1262 / Integrated production and purification of fibrinolytic protease from Mucor subtilissimus UCP 1262

As enzimas fibrinolíticas podem ser obtidas de micro-organismos por meio de processos fermentativos. O presente trabalho teve como objetivo avaliar a produção e extração integrada da protease fibrinolítica de Mucor subtilissimus UCP 1262 usando sistema de duas fases aquosas (SDFA). O processo integrado foi realizado para avaliar a produção, partição e recuperação da protease fibrinolítica, segundo planejamento experimental 23, utilizando como variáveis independentes a massa molar do polietileno glicol (PEG), a concentração do PEG e a concentração do sulfato de sódio. A maior atividade fibrinolítica (15,40U/mL) foi obtida na fase rica em sulfato de sódio no ensaio composto por 10% de sal e 18% de PEG 8000 (g/mol). Recuperações superiores a 80% foram obtidas. A protease fibrinolítica apresentou pH ótimo 7,0, estabilidade entre os pH 6,0 e 8,5, temperatura ótima 50°C, sendo estável de 10°C a 50°C. A enzima foi classificada como uma serino protease, com massa molecular de 52kDa. Como resultado, o processo é notavelmente eficaz para pré-purificar a protease fibrinolítica com baixo custo e rapidez significativa. Quando comparada a outras técnicas de produção e purificação isoladas, a fermentação extrativa é um processo digno a ser substituto das etapas iniciais de separação convencionais.(AU)

Fibrinolytic enzymes can be obtained from microorganisms through fermentative processes. The study aimed to evaluate the fibrinolytic protease production and integrated extraction from Mucor subtilissimus UCP 1262 by extractive fermentation using Aqueous Two-Phase Systems (ATPS). The integrated process was carried out to assess the production, partition and fibrinolytic enzyme recovery, according to a 2 3 -experimental design, using as independent variables Polyethylene glycol (PEG) molar mass, PEG and sodium sulphate concentration, concentration. The highest fibrinolytic activity (15.40U/mL) was obtained in sodium sulfate rich phase in the assay comprising of 10% of salt and 18% of PEG 8000 (g/mol). Yield greater than 80% was obtained. The fibrinolytic protease presented optimum pH 7.0 and stability between pH 6.0 and 8.5, and optimum temperature 50°C, stable between 10°C to 50°C. The enzyme was classified as a serine-protease with 52kDa of molecular weight. As a result, the process is remarkably effective to pre-purify the fibrinolytic protease with a low cost and significantly faster processing time. When compared to other isolated production and purification techniques the extractive fermentation is worthy of being a candidate to replace the initial stages of conventional separation processes.(AU)

Production and characterization of a biosurfactant produced by Streptomyces sp. DPUA 1559 isolated from lichens of the Amazon region

Surfactants are amphipathic compounds containing both hydrophilic and hydrophobic groups, capable to lower the surface or interfacial tension. Considering the advantages of the use of biosurfactants produced by microorganisms, the aim of this paper was to develop and characterize a biosurfactant produced by Streptomyces sp. DPUA1559 isolated from lichens of the Amazon region. The microorganism was cultured in a mineral medium containing 1% residual frying soybean oil as the carbon source. The kinetics of biosurfactant production was accompanied by reducing the surface tension of the culture medium from 60 to values around 27.14 mN/m, and by the emulsification index, which showed the efficiency of the biosurfactant as an emulsifier of hydrophobic compounds. The yield of the isolated biosurfactant was 1.74 g/L, in addition to the excellent capability of reducing the surface tension (25.34 mN/m), as observed from the central composite rotational design when the biosurfactant was produced at pH 8.5 at 28°C. The critical micelle concentration of the biosurfactant was determined as 0.01 g/mL. The biosurfactant showed thermal and pH stability regarding the surface tension reduction, and tolerance under high salt concentrations. The isolated biosurfactant showed no toxicity to the micro-crustacean Artemia salina, and to the seeds of lettuce (Lactuca sativa L.) and cabbage (Brassica oleracea L.). The biochemistry characterization of the biosurfactant showed a single protein band, an acid character and a molecular weight around 14.3 kDa, suggesting its glycoproteic nature. The results are promising for the industrial application of this new biosurfactant.

Produção e caracterização de protease fibrinolítica de Streptomyces parvulus DPUA 1573 / Production and characterization of fibrinolytic protease from Streptomyces parvulus DPUA 1573

As proteases fibrinolíticas são capazes de degradar coágulos de fibrina formados dentro dos vasos sanguíneos, evitando a trombose intravascular. Em animais, a tromboflebite, que acomete frequentemente os equinos, ocasiona, em seus casos graves, a obstrução jugular e também um edema de laringe, derivando a obstrução das vias aéreas, o que possibilita um edema cerebral, ocorrendo o óbito do animal. Devido ao fato de o tratamento ser de custo elevado, faz-se necessária a investigação de outras fontesde proteases fibrinolíticas com custos menores e com menos efeitos colaterais. Diante disso, este estudo tem como objetivo produzir e caracterizar proteases fibrinolíticas obtidas de Streptomyces parvulus DPUA 1573. Para produção da enzima, foi utilizado um planejamento fatorial 24 avaliando a concentração da farinha de soja (0,5, 1,0 e 1,5%) e da glicose (0, 0,5 e 1,0g/L), temperatura (28, 32 e 37ºC) e agitação (150, 200 e 250rpm) sobre a biomassa e a atividade fibrinolítica. Pode-se verificar que a protease fibrinolítica apresentou atividade máxima (835U/mL) nas condições de concentração de 1,5% de soja, 1g/L de glicose, 28°C e 150rpm com 48 horas de fermentação. A protease fibrinolítica obtida teve temperatura e pH ótimos de 55°C e pH 9,0, respectivamente. A atividade enzimática foi inibida pelo EDTA, pelo íon Fe2+ e pelo SDS, o que indicou a enzima ser uma metaloprotease. A linhagem Streptomyces parvulus DPUA 1573 foi capaz de produzir protease fibrinolítica, possuindo características bioquímicas favoráveis à aplicação na medicina veterinária e possivelmente humana.(AU)

Fibrinolytic proteases are able to degrade fibrin clot formed in the blood vessel, avoiding intravascular thrombosis. In animals, thrombophlebitis often affects horses, and in severe cases causes obstruction of the jugular and laryngeal edema leading to airway obstruction allowing cerebral edema resulting in the death of the animal. Since treatment is costly, the investigation of other sources of fibrinolytic proteases at lower cost and with fewer side effects is needed. Thus, this study aims to produce and characterize fibrinolytic proteases from Streptomyces parvulus DPUA 1573. For enzyme production, a factorial design was performed to evaluate 24 soybean flour concentration (0.5, 1.0 and 1.5%) and glucose (0, 0.5 and 1.0g/L), temperature (28, 32 and 37°C) and agitation (150, 200 and 250rpm) on biomass and fibrinolytic activity. Fibrinolytic protease showed maximum activity (835 U/mL) under these conditions: 1.5% soybean flour, 1g/L glucose, 28°C, and 150rpm 48 hours of fermentation. The optimal temperature was 55°C and optimal pH was 9.0. Fibrinolytic protease activity was inhibited by EDTA, the ion Fe2+, and by SDS, which indicated that the enzyme is a metallo-protease. The strain Streptomyces parvulus DPUA 1573 was able to produce fibrinolytic protease with biochemical characteristics favorable for application in veterinary and human medicine.(AU)

Produção de biocompostos com atividade antimicrobiana de Streptomyces sp. ante isolados de mastite caprina / Production of biocompounds with antimicrobial activity of Streptomyces sp. Isolated in face of goat mastitis

Actinomicetos são um dos principais produtores de enzimas, vitaminas e metabólitos secundários, destacando-se o gênero Streptomyces, o qual tem uma ampla capacidade de produção de antibióticos eficazes no combate a diferentes microrganismos, entre eles o Staphylococcus sp. Em virtude dessa eficiência no combate a patógenos, o objetivo deste trabalho foi avaliar a produção de metabólitos com atividade antimicrobiana produzidos por 67 Streptomyces isolados de liquens da região amazônica, ante isolados de mastite caprina (Staphylococcus aureus) do estado de Pernambuco, Brasil. Foi utilizado um planejamento fatorial para avaliar a influência das fontes de carbono (glicose) 0%, 0,5% e 1% e de nitrogênio (farinha de soja) 1%, 2,5% e 4% na produção dos antimicrobianos, bem como das variáveis pH, biomassa e atividade antimicrobiana. Dos Streptomyces estudados, o DPUA 1566 foi o que se destacou por formação de halos de inibição entre 18 e 26mm ante os isolados de mastite caprina. Foi possível verificar que a fonte de carbono inibiu a produção de antimicrobianos quando submetidos a uma concentração de glicose de 1%; com a retirada desta, os Streptomyces apresentaram uma elevada capacidade de produção de metabólitos com atividade antimicrobiana tendo potencial para o tratamento de mastite caprina.

Actinomycetes are a leading producer of enzymes, vitamins and secondary metabolites, especially the genus Streptomyces, which have a large capacity for the production of natural antibiotics and are effective against various micro-organisms including Staphylococcus sp. Due to this efficiency in combating micro-organisms, the aim of this study was to evaluate the production of metabolites with antimicrobial activity produced by Streptomyces isolated from lichens 67 in the Amazonia, compared to isolates from goat mastitis (Staphylococcus aureus) in the state of Pernambuco, Brazil. We used a complete factorial design to evaluate the influence of concentrations of carbon sources (glucose) at 0%, 0.5% and 1% and nitrogen (soybean flour) at 1%, 2.5% and 4% in the production of antimicrobial metabolites, and the influence of the pH, microbial biomass and activity variables. Of the studied Streptomyces DPUA 1566 what stood out was the formation of inhibition halos between 18 to 26 mm compared to the isolates from goats mastitis. It was noted that the carbon source inhibited the production of antimicrobial metabolites when subjected to a glucose concentration of 1%. However, after the discontinuation, Streptomyces showed a high capacity to produce metabolites with antimicrobial activity, which has an excellent potential for the treatment of mastitis in goats.

Avaliação da microbiota bacteriana do queijo de coalho artesanal produzido na região agreste do estado de Pernambuco / Evaluation of the bacterial microbiota of the artisanal "Coalho" of cheese produced in agreste region of the state of Pernambuco

O objetivo desta pesquisa foi avaliar a qualidade microbiológica e o perfil ácido-láctico do queijo de coalho artesanal. Todas as amostras de queijo apresentaram coliformes totais, termotolerantes e presença de Escherichia coli, porém com os valores dentro dos padrões estabelecidos pela legislação vigente no país. O perfil ácido-láctico estudado mostrou uma microbiota heterogênea, constituída por lactobacilos, lactococos, estreptococos e enterococos, confirmadas as espécies Enterococcus faecalis, Enterococcus faecium, Streptococcus thermophilus e Lactococcus lactis.

The aim of this study was to evaluate the microbiological quality and lactic-acid profile of artisanal coalho cheese. All cheese samples analyzed showed total coliforms, were thermotolerant, and had Escherichia coli, but all the values were within the standards established by current legislation in the country, and could be considered a food fit for human consumption. The cheese showed a heterogeneous microbiota, being constituted of all tested genus, such as lactobacilli, lactococcus, streptococcus and enterococcus, and confirmed the species: Enterococcus faecalis, Enterococcus faecium, Streptococcus thermophilus and Lactococcus lactis.

Pathogenic characteristics of yeasts isolated from vaginal secretion preserved under mineral oil

In order to evaluate the pathogenicity of yeasts isolated from vaginal secretion of pregnant and non-pregnant women - stored in mineral oil at the URM Mycology Collection, Department of Mycology, Federal University of Pernambuco - 30 samples belonging to the genera Candida, Rhodotorula, Trichosporon, and Kloeckera, were studied regarding their pathogenic characteristics, ability to grow at room temperature (28°C ± 1°C), 37°C, and 42°C for 72 hours, and production of both phospholipase and proteinase. Results showed that all 30 isolates (100%) were able to grow at room temperature and 37°C, and that 17 samples (57%) were able to grow at 42°C. Evaluation of enzymatic activity showed protease activity in only two isolates (7%), namely C. maritima and C. obtusa. Phospholipase activity was detected in 20 isolates (67%) using soy lecithin as substrate at different temperatures. The characterization of yeasts isolated from vaginal secretion and determination of their enzymatic activity may contribute to understanding the epidemiology of vulvovaginitis and assist in the treatment of patients.

Actinomicetos produtores de inibidores de beta-lactamases com atividade antimicrobiana frente a isolados de mastite bovina / Actinomycetes producers of beta-lactamases inhibitors with antimicrobial activity against microorganisms isolated from bovine mastitis

Avaliou-se a capacidade de 71 actinomicetos isolados de líquens da região amazônica em produzir inibidores de β-lactamases com atividade antimicrobiana sobre Staphylococcus aureus, resistentes à penicilina, isolados de mastite bovina do estado de Pernambuco. A seleção dos actinomicetos produtores de inibidores de β-lactamases foi realizada pela técnica de bloco de gelose contra Klebsiella pneumoniae ATCC 29665, e os actinomicetos selecionados foram testados frente a 17 linhagens de Staphylococcus aureus resistentes à penicilina. Os melhores produtores de inibidores de β-lactamases foram Streptomyces sp. DPUA 1542 e Nocardia sp. DPUA 1571, os quais foram submetidos ao cultivo submerso para determinação da curva de crescimento, pH e atividade antimicrobiana. Os maiores halos de inibição foram obtidos pelos metabólitos produzidos após 96 horas de cultivo tanto para Nocardia sp. - 13,5 e 12,0mm - como para Streptomyces sp. - 8,0 e 14,0mm - com os testes de difusão nos discos e poços, respectivamente. Os resultados permitiram concluir que os actinomicetos são fonte promissora de inibidores de β-lactamases, com potencial uso no tratamento de mastites bovinas.

The ability of 71 actinomycetes, isolated from the Amazon lichens, to produce β-lactamase inhibitors with antimicrobial activity was evaluated against penicillin-resistant Staphylococcus aureus, isolated from bovine mastitis in Pernambuco State. The selection of actinomycetes producers of β-lactamase inhibitors was performed using agar-plug method against Klebsiella pneumoniae ATCC 29665 and the selected actinomycetes were tested against 17 penicillin-resistant Staphylococcus aureus strains. The best producers of β-lactamase inhibitors were Streptomyces sp. DPUA 1542 and Nocardia sp. DPUA 1571. They were submitted to the submerged cultivation to determine the growth and pH curve, and antimicrobial activity. The highest inhibition halo zonewas obtained by metabolites produced after 96 hours of cultivation for both Nocardia sp. (13.5 and 12.0mm) and Streptomyces sp. (8.0 and 14.0mm) with discs and well diffusion tests, respectively. The results showed that the actinomycetes are a promising source of β-lactamase inhibitors, with potential for use in the bovine mastitis treatment.